Fig. 4

Changes in canine sperm acrosome status were detected by the CTC staining method during cold storage (5 °C) in the extenders containing different levels of melatonin versus the controls. A two-way repeated-measures ANOVA statistical test was used to make a comparison between treatment groups at different times. Pairwise comparisons with LSD confidence interval adjustments were carried out to examine the simple effects for the time and study groups. The different letters and numbers in the bar superscripts indicate a statistically significant difference (P ≤ 0.05) between study groups at the specified time point and between time points at the specified group, respectively. Negative Control (without Melatonin or DMSO), Positive Control (containing DMSO), M1 (containing 0.5 mM Melatonin), M2 (containing 1 mM Melatonin), and M3 (containing 2 mM Melatonin), ns, non-significant. All numbers are presented as Means±SEM. Pattern F = non-capacitated with intact acrosome, pattern B = capacitated with intact acrosome, and Pattern AR = reacted/damaged acrosome, CTC=Chlortetracycline